The major goal of the Project Head's laboratory is to understand the mechanisms by which homozygous sickle cell (SS) red blood cells (RBCs) become irreversible sickled cells (ISCs) and dense; a step towards devising therapeutics that block formationof ISCs and cellular dehydration. We have demonstrated that ISC membrane skeletons disassemble far more slowly at 37 degrees C than reversible sickled cells (RSCs) and control membrane skeletons. This suggested that the reason that ISCs cannot change shape is because their membrane skeleton cannot dynamically disassemble and reassemble. Furthermore, we demonstrated that this slow disassembly was clue to modifications in spectrin and actin. The modification in ISC beta-actin has been demonstrated to be a C284-C373 disulfide bridge which leads to actin filaments that slowly disassemble. We do not yet know the modification of ISC spectrin that leads to a slower disassociation of the spectrin-4.1-actin ternary complex. Spectrin is an E2 ubiquitin conjugating enzyme as well as a target for ubiquitin.The E2 thioester site and one target site are within spectrin repeats alpha 20/21. Interestingly, ubiquitination of SS spectrin in its DTT sensitive E2 site and DTT insensitive target sites is reduced by 80 to 90% probably due to glutathiolation of the E2 cysteine. Since the alpha 20/21 repeat is the heterodimer nucleation site and associated with the protein 4.1 and adducin sites on beta-spectrin, we hypothesize that lack of ubiquitination of SS spectrin would lead to faster rates of heterodimer formation and higher affinity spectrin-4.1-actin and spectrin-adducin-actin ternary complex formation. This would supply an answer to why the ISC skeleton dissociates far more slowly than the RSC or control skeleton. In this proposal we will identify the precise cysteines and lysines involved in E2 (and possibly E3) and target sites of alpha 20/21 respectively; utilizing the proteomics technology of isotope coded affinity tags and LC-MS/MS (Aim 1). We will then determine the role of spectrin ubiquitination in regulating heterodimer formation (Aim 2) and spectrin-4.1-actin and spectrin-adducin-actin formation and disassembly (Aim 3).